| The integrated rice-fishing model is an important part of my country’s traditional ecological agriculture,and the Qingtian rice-fish symbiosis system is an outstanding representative of this model.The Qingtian rice-fish symbiosis system has a long history.In 2005,the Food and Agriculture Organization of the United Nations(FAO)included it in the first batch of Globally Important Agricultural Heritage Systems(GIAHS,Globally Important Agricultural Heritage Systems)protection list,and received global attention.Cyprinus carpio var.qingtianensis is the core of the system,and its original species protection is also an important part of agricultural cultural genetic protection.After a long period of artificial selection and adaptive evolution in shallow water environment,Cyprinus carpio var.qingtianensis has formed a unique "Jahwa" artificial breeding species,which has very high application value and research value.A major feature of the Qingtian rice-fish symbiosis system is that its paddy field water environment is only 20 cm deep,the overall dissolved oxygen level is low and the daily variation range is large,but there are few research reports on how Cyprinus carpio var.qingtianensis adapt to such a special low-oxygen water environment.Based on the parameters of Cyprinus carpio var.qingtianensis tolerance to hypoxia and the daily changes of dissolved oxygen in the local paddy field water environment,this study simulated acute hypoxia and reoxygenation environment,analyzed and screened out important pathways with the help of transcriptome and metabolomics,and finally used Real-time PCR detects the expression levels of differential genes in key tissues of Cyprinus carpio var.qingtianensis after acute hypoxia stress and reoxygenation recovery treatments,and studies its physiological regulation under acute dissolved oxygen changes from the perspectives of biological processes and molecular mechanisms The mechanism also provides a reference for similar studies of other fish.The main findings are as follows:1.Comparison of the brain transcription characteristics of Cyprinus carpio var.qingtianensis after acute hypoxia and reoxygenation.The results showed that 1094 differentially expressed genes(DEGs)were screened out in hypoxia group and normoxia group,including 730 up-regulated and 364 down regulated.After KEGG enrichment analysis,it is shown that DEGs are involved in processes such as apoptosis,inflammation,and energy metabolism.The analysis found that the anaerobic energy-releasing glycolytic pathway was enhanced during hypoxia stress,producing a large amount of NADH and pyruvate,which provided a large amount of sugar raw materials for the gluconeogenesis pathway.In the process of hypoxia stress,the pentose phosphate pathway is activated,and its final products ribose and NADPH not only provide raw materials for the transcription process,but also provide energy for metabolism and reduce the body’s oxidative damage.After reoxygenation,there were 2288 DEGs,803 of which were up-regulated and 1485 down regulated.KEGG enrichment analysis showed that DEGs were involved in gluconeogenesis,regulation of cell differentiation and growth,lipid metabolism and reduction of apoptosis.KEGG enrichment analysis showed that DEGs were involved in gluconeogenesis,regulating cell differentiation and growth,lipid metabolism and attenuating cell apoptosis.These differentially expressed genes can effectively maintain circulating blood glucose levels in the body,alleviate acidosis,and stabilize the normal differentiation and growth of cells.2.Comparison of characteristics of liver and pancreas metabolome of Cyprinus carpio var.qingtianensis after acute hypoxia stress and reoxygenation recoveryA total of 85 metabolites were screened out from the comparative analysis of hypoxia stress 6h group and normoxia group,including 46 up-regulated and 39 down regulated.These differential metabolites are mainly involved in the regulation of protein,lipid and purine metabolism,suggesting that the metabolism of carbohydrate will be weakened and the glycolysis pathway will be enhanced during hypoxia.The transamination role of amino acid metabolism plays an important role in the metabolism of Qingtian fish In particular,glutamic acid and glutamine metabolism can effectively eliminate ammonia toxicity and complete energy supply.A total of 147 metabolites were screened out in the comparison of 6 h reoxygenation repair group and 6 h hypoxia stress group,of which 53 were up-regulated and 94 were down regulated.According to KEGG pathway enrichment analysis,it was speculated that the gluconeogenesis involved in glutamate and glutamine was weakened,and the transamination of amino acid metabolism was inhibited.The difference between transcriptome and metabolome in the expression level of glucose metabolism during reoxygenation may be due to the fact that metabolome data reflect the physiological metabolic process.Besides enzymes,many enzymatic reactions are reversible,and the substrate concentration determines the direction of biochemical reactions.There is a blood-brain barrier in brain tissue.Glucose can not directly pass through the blood-brain barrier.Ketosome and lipid metabolism may replace gluconeogenesis.3.Cloning of vegf-a gene and restoration of tissue expression under hypoxia stress and reoxygenationThe g DNA sequence of vegf-a is 2821 bp in length,including an open reading frame with 5’UTR of 680 bp and 3’UTR of 1721 bp,encoding a total of 139 amino acids;the sequence alignment results show that the encoded amino acid sequence is similar to that of the carp(Cyprinus carpio)vegf-a encodes a high homology,and clusters into one branch on the phylogenetic tree;q RT-PCR results show that vegf-a is expressed in all tissues of Cyprinus carpio var.qingtianensis,among which the hepatopancreas and white muscle The relative expression level is higher,and the heart and head kidney are less;after 6 hours of hypoxia treatment and 6 hours of reoxygenation recovery,the expression of vegf-a gene in the gills,white muscles and hepatopancreas has been showing a significant upward trend(P< 0.05),the expression level of other tissues increased first(P<0.05),and then decreased.This indicates that the different tissues of Qingtiantian fish have different sensitivity to oxygen,and there are differences in the expression pattern of vegf-a.In the process of hypoxia stress and reoxygenation recovery,vegf-a participates in the adaptive physiological regulation of Qingtiantian fish to acute dissolved oxygen changes by regulating the rate of angiogenesis. |
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